The present invention relates to methods for effective targeting of mRNA in vivo in a subject, by oral administration of a nuclease-resistant morpholino antisense compound having uncharged phosphorus-containing backbone linkages. The invention further relates to methods and kits for detection of base-specific intracellular binding events involving one or more target RNAs in a subject, wherein a heteroduplex comprising a target RNA and an antisense oligomer is detected in a body-fluid sample taken from the subject.
Many diseases and other medical conditions are characterized by the presence of undesired DNA or RNA, which may be in certain instances single stranded and in other instances in double stranded. These diseases and conditions can be treated using the principles of antisense therapy which includes targeting a specific DNA or RNA target sequence through complementarity or through another specific binding means.
In therapeutic applications, antisense oligomers formulated for oral administration have met with limited success, which in general is attributed to the effects ubiquitous nucleases present in vivo and to the overall charge of the molecules which limits their ability to get into cells.
Numerous references directed to enhanced oral delivery of such antisense oligonucleotides for the diagnosis and treatment of disease may be found in the literature.
Diagnosis and monitoring of various disease conditions is accomplished by an analysis of peptides, proteins, antibodies or nucleic acids associated with the condition.
Genetic analysis of individuals usually focuses on two types of information: The first is analysis of mutations associated with various genetic diseases, e.g., cystic fibrosis, Huntington""s disease, and certain cancers known to be associated with genetic mutations, e.g., breast cancer. The second involves the analysis of relative levels of expression of genes under certain conditions,. e.g., in response to drug treatment, in various diseases or conditions, or differences in levels of gene expression among different tissues.
Currently, genetic analyses of this type are carried out ex vivo, typically by obtaining a tissue of blood sample from an individual, and analyzing genomic DNA, cDNA or mRNA for the presence of absence of certain mutations or for elevated or depressed levels of gene expression. Diagnostic devices, e.g., gene chips, for detecting mutations or changes in level of expression are now available, and with new capabilities under development.
Similar methods may be employed to monitor the effect of therapeutic compounds on gene expression in individuals. That is, following compound administration, a tissue biopsy or blood sample may be obtained from the treated patient to determining the effect of the compound on expression of one or more targeted genes.
Although analysis of mutations and levels of gene expression by these in vivo methods has the capability of yielding important information about gene makeup and drug response in an individual, the methods are many times impractical, expensive and/or unable to provide the desired information. For example, it is generally not practical to biopsy an individual""s tissue to monitor gene expression, both because of the difficulty and risk to patient of obtaining a tissue sample, and because of the expense of working up a tissue sample for analysis.
It would therefore be highly desirable to be able to target gene mutations and monitor levels of gene expression, or gene expression in response to therapeutic agents by methods that do not require obtaining tissue or cellular samples from an individual, nor and isolating and measuring nucleic acids samples obtained from such cells or tissue.
The present invention addresses a deficiency in the prior art by providing a method for effective targeting of mRNA in vivo in a subject, by oral administration of a nuclease-resistant morpholino antisense compound having uncharged phosphorus-containing backbone linkages in a manner which results in modulated expression of the gene product encoded by the mRNA target.
In one aspect, the antisense compound preferably has a length of about 8 to 40 bases, more preferably 12 to 25 bases.
In another aspect, the antisense compound preferably has intersubunit linkages selected from the group consisting of the structures presented in FIGS. 2A-2E, and exemplified particularly by the phosphorodiamidate linkage represented at FIG. 2B, where X=NH2, Y=O, and Z=O.
In an further aspect, the morpholino antisense compound contains a targeting base sequence that is complementary to a region that spans the translational start codon of a selected target gene.
A preferred target gene for an orally administered antisense oligomer of the invention is a gene associated with a proliferative disorder, e.g., cancer. An exemplary sequence is the one identified by SEQ ID NO: 2.
In a related aspect, the invention provides a method of detecting, the occurrence of a base-specific intracellular binding event involving a target RNA in a subject, by performing the steps of (a) administering to the subject a morpholino antisense compound including a targeting base sequence that is complementary to a region that spans the start codon of a selected gene, and uncharged, phosphorous-containing intersubunit linkages, in an amount effective to hybridize to a region of the target RNA with a Tm substantially greater than 37xc2x0 C., (b) taking a sample of a body fluid from the subject at a selected time after administering the oligonucleotide, and (c) detecting the presence of a nuclease-resistant heteroduplex composed of the antisense oligonucleotide and the target RNA region in the sample.
In practicing the method, it is preferred that the antisense oligomer have a length of about 8 to 40 bases and be effective following oral administration.
The methods of the invention provide for detection of antisense oligomer:RNA heteroduplexes in a body fluid such as urine, saliva, plasma, or blood, preferably urine.
In one aspect, detection of the heteroduplex is accomplished by reacting the sample with an antibody specific for the heteroduplex, and detecting the presence of the antibody-heteroduplex conjugate.
In other cases, the methods of the invention provide an antisense oligomer comprising a reporter molecule such that detection of antisense oligomer:RNA heteroduplexes is accomplished by detecting the presence of the reporter molecule associated with the heteroduplexes.
The invention further provides diagnostic methods for assessing the biological condition of a subject, wherein the biological condition is associated with expression of a target gene and the methods are used to detect changes in RNA associated with expression of the target gene.
In one preferred application, the method may be used to detect changes in expression of a target gene in response to a therapeutic agent in the subject, wherein the target RNA is mRNA produced by expression of a selected gene, e.g., a gene associated with a known disease state.
The invention is applicable to single or multiple administrations of nuclease-resistant antisense oligomers for therapeutic and or diagnostic applications.
The invention also provides kits for detecting the occurrence of a base-specific intracellular binding event involving a target RNA in a subject comprising a nuclease-resistant antisense oligomer complementary to a target RNA which hybridizes to the target with a Tm substantially greater than 37xc2x0 C., and a means for detecting a heteroduplex formed between the antisense oligomer and the target RNA.
These and other objects and features of the invention will become more fully apparent when the following detailed description is read in conjunction with the accompanying drawings.